PROJECT SUMMARY Through three inter-related and inter-dependent Projects and four essential Cores, our team will continue to define the biological basis of dysfunctional sphingolipid metabolism in AML, and in that process, validate new therapeutic targets for pharmacological treatment approaches. The premise of the renewal P01 application is that targeting enzymes responsible for dysfunctional sphingolipid metabolism will lead to new clinical options in AML. The overall hypothesis to be tested by all projects is that increasing endogenous pro-apoptotic ceramide species, while diminishing pro-survival phosphorylated or glycosylated ceramide metabolites, will yield efficacious treatments for AML. The overall premise of this renewal application are the exciting results from the first-in-man clinical trial of ceramide nanoliposomes for solid tumors (NCT02834611), demonstrating the lack of dose-limiting toxicities at doses where stable disease is observed. Exciting preliminary and published in vivo data provide the rationale for nanoscale delivery vehicles for exogenous ceramide as an adjuvant therapy to support standard of care therapy (low-dose AraC and venetoclax) in a phase Ib/IIa clinical trial for relapsed/refractory AML (Project 1, CAV trial, UVA Protocol Review Committee approval #5414, pre-IND 142902). In addition, preliminary and published data are provided demonstrating that selective inhibitors of ceramide metabolism (Projects 2-3) increase the clinical utility of agents that raise levels of pro-apoptotic ceramides. A common scientific theme of all Projects is the mechanistic investigation of drug resistance and programmed cell death, which can be directly altered with sphingolipid-based therapeutics. The major innovation of our P01 is to utilize a bioinformatic and systems biology approach to integrate genomics, sphingolipidomics, and proteomics data from molecularly defined patient samples to reveal susceptible populations for testing innovative sphingolipid-targeted therapeutics in state-of-the-art patient-derived xenografts, genetically engineered murine models, and most importantly, a clinical trial. The major goal of this proposal is to develop new sphingolipid-targeted therapeutics for AML. The interdisciplinary team includes leaders in the fields of leukemia (specifically AML), nanoscale delivery systems for bioactive lipids, programmed cell death, and the biochemistry and biophysics of sphingolipids. This major goal will be accomplished through the following overarching five Specific Aims that are shared by all Projects and Cores: 1) Evaluate the efficacy of therapeutics that elevate exogenous or endogenous levels of pro-apoptotic ceramide species in animal and clinical trials; 2) Obtain preclinical and clinical PK, bio-distribution, and toxicology data to support and/or expand our FDA-IND for sphingolipid-based AML therapeutics; 3) Define the biochemical, biophysical, and molecular mechanisms underlying the synergies obtained with agents that target dysfunctional sphingolipid metabolism; 4) Understand the molecular basis defining variable dysfunctional sphingolipid metabolism for heterogeneous AML; 5) Define and validate lipid-based biomarkers as diagnostic or prognostic indicators.